The long-term goal of this research program is to understand at the molecular level the cellular mechanisms of heart failure. Myocardial ischemia is associated with changes in calcium flux, pH, and enzyme activities. A major goal is to determine the structure of Na+,K+-ATPase and the mechanism of action digitalis. The complete primary sequence of the alpha subunit has been determined via the cDNA route. Several labeled peptides representing important domains have been isolated by HPLC. The FITC peptide from a variety of ATPase sources has been sequenced. There is a remarkable homology between the Na,K-ATPase and the S.R. Ca-ATPase. The next step is to obtain the the sequence of the beta subunit. Further structural studies are planned. Both the low capacity/high affinity and high capacity/low affinity ouabain binding sites are reduced ca. 30% in one-kidney Goldblatt model of hypertension. Pharmacological and radioligand binding studies indicate that both high and low affinity sites for the dihydropyridines exist in membranes from coronary arteries and heart. Methods have been developed to study calcium binding to TNC and tension development in skinned skeletal muscle fibers. A phosphatase capable of dephosphorylating myosin and modifying the interaction between actin and myosin has been isolated from mammalian vasulat smooth muscle and is being studied. There is preliminary evidence that TNA may exist in both adult and embryonic forms. It will be determined if the embryonic TNI is responsible for a lack of effect of acidic pH on Ca-activation of immature cardiac myofibrils. The mechanism by which cAMP-dependent and Ca-calmodulin-dependent protein kinases regulate calcium transport processes in S.R. will be investigated. The role of the two distinct forms of ADP sensitive phosphorylated intermediates of rabbit skeletal muscle will be studied. Clonazepam will be used to elucidate the molecular nature and role of Na-Ca exchange of mitochondria in cardia muscle. Mixed monolayers of trioleoylglcerol and egg phosphatidylchloline have been used to study interfacial catalsis of lipolytic enzymes. These results show that the rates of catalysis are dependent upon the neutral lipids surface pressure and apolipoprotein C-II. These findings will be explored in depth.